1. Field of the Invention
This invention relates to the purification of N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester (neotame) by crystallization to give highly pure product with high yields.
2. Related Background Art
N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester (neotame) is a high potency dipeptide sweetener (about 8000xc3x97 sweeter than sucrose) that has the formula 
The chemical synthesis of neotame is disclosed in U.S. Pat. Nos. 5,480,668, 5,510,508 and 5,728,862, the disclosure of each of which is incorporated by reference herein. These chemical processes produce several troublesome impurities, including N,N-di(3,3-dimethylbutyl)-L-aspartyl-L-phenylalanine methyl ester (dialkylated aspartame), xcex1-methyl hydrogen-3-(3,3-dimethylbutyl)-2-L-(2,2-dimethylpropyl)-5-oxo-xcex1-L-(phenylmethyl)-1,4(L)-imidazolidine diacetate (dialkylated imidazolidinone), N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine (demethylated xcex1- or xcex2-neotame) and methyl ester of N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester (methylated xcex1- or xcex2-neotame). These impurities are represented respectively by the structural formulae: 
U.S. Pat. No. 5,728,862 outlines a purification method by which neotame is precipitated out of an aqueous/organic solvent solution, wherein the aqueous/organic solvent solution has an amount of organic solvent of about 17% to about 30% by weight.
Since neotame is mainly employed in foods for human consumption, it is extremely important that neotame exist in a highly purified state. Thus, it is clear that there is a need to economically produce pure N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester.
The present invention is directed to a process of purifying N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester by crystallization from an aqueous solvent comprising the steps of: distilling organic solvent from an N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester reaction mixture; adding water to the reaction mixture to reach a desired organic solvent concentration; holding the reaction mixture for a time and at a temperature sufficient to hydrolyze dialkylated imidazolidinone to neotame and 3,3-dimethylbutyraldehyde; optionally, seeding the reaction mixture at an appropriate temperature and with an amount of N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester; and cooling the reaction mixture in order to crystallize N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester.
Another embodiment of the present invention is directed to a process of purifying N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester by crystallization from aqueous solvent comprising the steps of: adding water to an N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester reaction mixture; holding the reaction mixture for a time and at a temperature sufficient to hydrolyze dialkylated imidazolidinone to neotame and 3,3-dimethylbutyraldehyde; and distilling organic solvent in order to reach a desired organic solvent concentration and crystallize N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester.
Yet another embodiment of this invention is directed to a process for purifying N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester from an organic solvent comprising the steps of: dissolving N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester in an organic solvent; and crystallizing the purified N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester from the organic solvent. In this embodiment, the use of water is not required. Of course, the organic solvent may be, and preferably is, a mixture of organic solvents. For example, a first organic solvent may be added to dissolve N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester and a second organic solvent may be added to crystallize N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester.
A further embodiment of the invention is directed to a process for purifying N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester by simply mixing N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester with water and holding the mixture for a time and at a temperature sufficient to hydrolyze dialkylated imidazolidinone. The mixture may be completely aqueous, but preferably includes an organic solvent, which most preferably is methanol. The N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester may be isolated by any manner, e.g., crystallization or chromatographic separation, but most preferably by crystallization.
In preferred embodiments of the present invention, the organic solvent is selected from methanol, ethanol, isopropanol, butanol, ethyl acetate, methyl acetate, propyl acetate, acetone, tetrahydrofuran, acetonitrile, dichloromethane, toluene, cyclohexane, hexane, heptane, tertiary butyl methyl ether, diethyl ether, methyl ethyl ketone and mixtures thereof. In a particularly preferred embodiment of the present invention, the solvent is methanol.
According to the present invention, crystallization of N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester from the reaction mixture can be optimized in order to produce substantially pure N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester.
It has been found that N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester crystallization in aqueous methanol has a wide meta-stable crystallization zone which inhibits rapid nucleation. Seeding during crystallization can initiate a controlled crystal growth rate according to the present invention. Regardless, the same type of monohydrate crystals are obtained over a wide range of conditions (e.g., static, stirred, evaporative, slow cooling, and precipitation by non-polar solvent addition).
There are two methods by which the crystallization of the present invention can be performed. The first method is referred to as slow cooling. The second method is referred to as evaporative crystallization.
According to the first embodiment of the present invention, an organic solvent, such as methanol, is distilled from a reaction solution, and water is added. Then, the solution is held for a time and at a temperature sufficient to hydrolyze dialkylated imidazolidinone to a-neotame and 3,3-dimethylbutyraldehyde. The solution is optionally seeded. Finally, the solution is slowly cooled to crystallize N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester.
The reaction mixture, if seeded, is seeded in an amount from 0.0001%-10%, by weight of the N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester in the solution. Preferably this amount is from 0.1% to 1%. Most preferably this amount is from 0.1% to 0.5%. Seeding can occur between 5xc2x0 C. and 300xc2x0 C., preferably between 25xc2x0 C.-30xc2x0 C.
According to this method, the reaction mixture is generally cooled to a temperature of from about 5xc2x0 C. to about 25xc2x0 C. Crystallization preferably occurs by ramping the temperature after seeding to 4xc2x0 C. over a time range of 0.5 to 12 hours.
Generally, lower seed and faster ramp gives larger crystals and slightly higher yields. Lesser seed also generally gives more uniformly sized crystals.
According to the second embodiment of the present invention, water is added to the reaction mixture, the solution is held for a time and at a temperature sufficient to hydrolyze dialkylated imidazolidinone to a-neotame and 3,3-dimethylbutyraldehyde and then an organic solvent, such as methanol, is distilled to the desired concentration. The N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester crystallizes during the distillation of the organic solvent. The solution may be optionally seeded as described above.
An additional embodiment of the present invention comprises adding water to the reaction mixture and then distilling an organic solvent, such as methanol, to the desired concentration. During this process, the dialkylated imidazolidinone is hydrolyzed to xcex1-neotame and 3,3-dimethylbutyraldehyde. Again, the N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester crystallizes during the distillation of the organic solvent, and the solution may be optionally seeded as described above.
Additional embodiments of the present invention include purifying N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester by mixing it with water or with water and an organic solvent and holding the solution to hydrolyze dialkylated imidazolidinone to xcex1-neotame and 3,3-dimethylbutyraldehyde.
As noted previously, methanol is the preferred organic solvent. According to the present invention, crystals grow satisfactorily from seeds in both 15% and 35% methanol solutions, with higher methanol concentrations requiring a lower temperature for crystal growth to accelerate. It should also be noted that higher methanol concentration during crystallization at lower temperatures results in higher purity neotame in high yield.
In additional embodiments of the present invention, organic solvents other than methanol may be used to crystallize N-[N-(3,3-dimethylbutyl)-L-xcex1-aspartyl]-L-phenylalanine 1-methyl ester. Such alternate organic solvents include, without limitation, ethanol, isopropanol, butanol, ethyl acetate, methyl acetate, propyl acetate, acetone, tetrahydrofuran, acetonitrile, dichloromethane, toluene, cyclohexane, hexane, heptane, tertiary butyl methyl ether, diethyl ether, methyl ethyl ketone and mixtures thereof.
According to the present invention, the desired solvent concentration is generally between 15-35% by weight.
In a preferred embodiment of the present invention, the desired solvent concentration is between 25-30% by weight.
Further, in accordance with the present invention, the reaction mixture is held in order to hydrolyze the dialkylated imidazolidinone to a-neotame and 3,3-dimethylbutyraldehyde. The reaction mixture is generally held for 2-20 hours at a temperature of 40xc2x0 C.-45xc2x0 C. In a preferred embodiment of the present invention, the reaction mixture is held for 4-9 hours.
The reaction mixture or the solution containing neotame may be unstirred or stirred during the crystallization processes of the present invention.
The product isolated from any of these crystallization methods is the same monohydrate, which may be dried to produce an anhydrous form. The monohydrate formation is independent of cooling ramp, seed amount, seeding temperature and final temperature.
The Examples which follow are intended as an illustration of certain preferred embodiments of the invention, and no limitation of the invention is implied.